Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease
Authors: Junping Yin, Qi Mei, Marco Prinz, Zeinab Abdullah, Ulf Panzer, Jian Li, Sibylle von Vietinghoff, Christian Kurts
Journal: Kidney International (2023)

Renal mononuclear phagocytes (MNPs), including macrophages and dendritic cells, form a contiguous sentinel network throughout the kidney interstitium and participate in maintaining homeostasis and the defense against pathogens.1Kurts C. Panzer U. Anders H.J. et al.The immune system and kidney disease: basic concepts and clinical implications.Nat Rev Immunol. 2013; 13: 738-753Crossref PubMed Scopus (460) Google Scholar,2Liu F. Dai S. Feng D. et al.Distinct fate, dynamics and niches of renal macrophages of bone marrow or embryonic origins.Nat Commun. 2020; 11: 2280Crossref PubMed Scopus (37) Google Scholar MNPs are also involved in inflammatory kidney disease (e.g., in crescentic glomerulonephritis and inflammasome-mediated tissue fibrosis).1Kurts C. Panzer U. Anders H.J. et al.The immune system and kidney disease: basic concepts and clinical implications.Nat Rev Immunol. 2013; 13: 738-753Crossref PubMed Scopus (460) Google Scholar Accumulation of MNPs is a prominent feature in nephritis,3Anders H.J. Vielhauer V. Schlöndorff D. Chemokines and chemokine receptors are involved in the resolution or progression of renal disease.Kidney Int. 2003; 63: 401-415Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar and reducing MNP numbers is protective in most nephritis models.3Anders H.J. Vielhauer V. Schlöndorff D. Chemokines and chemokine receptors are involved in the resolution or progression of renal disease.Kidney Int. 2003; 63: 401-415Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar The recruitment of circulating monocytes contributes to MNP accumulation, and its inhibition (e.g., by blocking chemokines or their receptors) ameliorates nephritis partially.2Liu F. Dai S. Feng D. et al.Distinct fate, dynamics and niches of renal macrophages of bone marrow or embryonic origins.Nat Commun. 2020; 11: 2280Crossref PubMed Scopus (37) Google Scholar Also, local proliferation of tissue-resident MNPs might theoretically contribute to their accumulation in nephritis. This would impact therapeutic approaches to treat nephritis by preventing the recruitment of MNP precursors. Most reporter mice used for studying MNPs express a single fluorescence molecule in the whole MNP population and, hence, cannot discriminate individual cells. Such discrimination has become possible with Confetti multicolor fluorescence reporter mice that stochastically express different fluorochromes in distinct cell types after crossing to suitable CRE-transgenic mice. In nephrology, these mice have been used to differentially label kidney-resident cells, like podocytes, tubular epithelial cells, or renin-lineage cells.4Tao J. Polumbo C. Reidy K. et al.A multicolor podocyte reporter highlights heterogeneous podocyte changes in focal segmental glomerulosclerosis.Kidney Int. 2014; 85: 972-980Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar For motile cells, such as renal MNPs, this technique also allows distinguishing between recruitment and local proliferation, because the former results in homogenic distribution of same-color MNPs, whereas local proliferation generates clones of the same fluorescence color. A suitable tool is the Microfetti mouse line, generated by crossing Confetti mice with CX3CR1-CRE mice, in which microglia cells randomly expressed 1 of 4 fluorescent proteins: green fluorescent protein (GFP), yellow fluorescent protein, red fluorescent protein, and cyan fluorescent protein.5Masuda T. Amann L. Monaco G. et al.Specification of CNS macrophage subsets occurs postnatally in defined niches.Nature. 2022; 604: 740-748Crossref PubMed Scopus (37) Google Scholar,6Tay T.L. Mai D. Dautzenberg J. et al.A new fate mapping system reveals context-dependent random or clonal expansion of microglia.Nat Neurosci. 2017; 20: 793-803Crossref PubMed Scopus (345) Google Scholar We previously showed that pathogenic renal MNPs in nephrotoxic nephritis (NTN) express and depend on CX3CR1.7Hochheiser K. Heuser C. Krause T.A. et al.Exclusive CX3CR1 dependence of kidney DCs impacts glomerulonephritis progression.J Clin Invest. 2013; 123: 4242-4254Crossref PubMed Scopus (79) Google Scholar Thus, Microfetti mice allowed clarifying whether these MNPs locally proliferate in nephritis models. To label renal MNPs, a single dose of tamoxifen was injected s.c. into Microfetti mice, as described5Masuda T. Amann L. Monaco G. et al.Specification of CNS macrophage subsets occurs postnatally in defined niches.Nature. 2022; 604: 740-748Crossref PubMed Scopus (37) Google Scholar,6Tay T.L. Mai D. Dautzenberg J. et al.A new fate mapping system reveals context-dependent random or clonal expansion of microglia.Nat Neurosci. 2017; 20: 793-803Crossref PubMed Scopus (345) Google Scholar (Figure 1a and Supplementary Methods). One week later, Confetti-labeled cells were evident in the renal cortex and medulla (Figure 1b; Supplementary Figure S1). Costaining with F4/80 confirmed that Confetti+ cells were MNPs (Figure 1a and b). Each individual Confetti+ MNP expressed 1 of the 4 fluorescent proteins in different cellular compartments: GFP in the nucleus, cyan fluorescent protein in the cell membrane, yellow fluorescent protein in the cytoplasm, and red fluorescent protein in the cytoplasm (Supplementary Figure S2A and B). No labeling was seen in Microfetti mice not injected with tamoxifen, and proliferating MNPs were not preferentially labeled (data not shown), consistent with previous findings.6Tay T.L. Mai D. Dautzenberg J. et al.A new fate mapping system reveals context-dependent random or clonal expansion of microglia.Nat Neurosci. 2017; 20: 793-803Crossref PubMed Scopus (345) Google Scholar This indicated that stochastic multicolor labeling of renal MNPs was possible by using Microfetti mice. To study clonotypic labeling in models of glomerulonephritis and tubulointerstitial nephritis, we next labeled MNP in mice with NTN7Hochheiser K. Heuser C. Krause T.A. et al.Exclusive CX3CR1 dependence of kidney DCs impacts glomerulonephritis progression.J Clin Invest. 2013; 123: 4242-4254Crossref PubMed Scopus (79) Google Scholar or under high adenine diet (HAD),8Ludwig-Portugall I. Bartok E. Dhana E. et al.An NLRP3-specific inflammasome inhibitor attenuates crystal-induced kidney fibrosis in mice.Kidney Int. 2016; 90: 525-539Abstract Full Text Full Text PDF PubMed Google Scholar respectively. Healthy Microfetti mice were used as a control and showed a homogenic distribution of Confetti colors (Figure 1c and d). One week after NTN or 10 days after HAD induction, kidneys were collected for analysis (Figure 1e and g). Immunofluorescence microscopy revealed clustering of Confetti+ cells in the renal cortex of NTN mice and in the medulla of mice under HAD (Figure 1f and h). Each of these clusters expressed only 1 of the 4 Confetti colors, identifying them as clonally expanded cells. Using this setup, we analyzed the distribution and relationship of Confetti+ MNPs by generating a rendering model on the maximum-intensity projection of the confocal stack for computational analysis (Figure 2a). Our algorithm measured the proximities of each individual Confetti+ MNP to each same-color MNP and compared them with Monte Carlo–simulated data, where MNPs are assigned random colors, resulting in random distances (Figure 2a). We defined the recorded data that lay above the 98% of the Monte Carlo data points as a nonrandom distribution of same-color MNPs and considered them progeny of clonal proliferation6Tay T.L. Mai D. Dautzenberg J. et al.A new fate mapping system reveals context-dependent random or clonal expansion of microglia.Nat Neurosci. 2017; 20: 793-803Crossref PubMed Scopus (345) Google Scholar (Figure 2b). This procedure allowed inferring whether the distribution of adjacent same-color MNPs was due to clonal expansion or random chance. We next quantified clonal expansion of renal MNPs under disease conditions. First, we generated rendering models of renal cortex and medulla from healthy mice, NTN mice, and HAD mice (Supplementary Figure S3). The density of Confetti+ MNPs in these models was subsequently calculated using the above-mentioned algorithm. In mice with NTN, we observed around 4 division events, meaning 4 same-color Confetti+ MNPs derived from the same parent cell, in the cortex (Figure 2c and d), but not in the medulla (Figure 2e and f), consistent with the cortical location of inflamed glomeruli in glomerulonephritis. By contrast, mice under HAD showed MNP division events exclusively in the medulla (Figure 2c–f), where osmolarity is higher and adenine crystals are more likely to precipitate than in the cortex. No division event was seen in healthy mice (Figure 2c–f). A total of 0.5% to 1% of blood monocytes were Confetti+ at 1 week after tamoxifen injection (Supplementary Figure S4), consistent with previous reports in central nervous system disease models.5Masuda T. Amann L. Monaco G. et al.Specification of CNS macrophage subsets occurs postnatally in defined niches.Nature. 2022; 604: 740-748Crossref PubMed Scopus (37) Google Scholar,6Tay T.L. Mai D. Dautzenberg J. et al.A new fate mapping system reveals context-dependent random or clonal expansion of microglia.Nat Neurosci. 2017; 20: 793-803Crossref PubMed Scopus (345) Google Scholar This was much lower than the 25% to 30% of renal MNPs carrying the label (Supplementary Figure S4), but we nevertheless wanted to verify that Confetti+ MNPs were derived from tissue-resident MPs. To this end, we treated Microfetti mice with tamoxifen and induced kidney disease after 8 weeks. At this late time point, hardly any monocytes retained the label, whereas still >20% of renal MNPs did so (Supplementary Figure S4). By utilizing the above-mentioned algorithm, the densities of Confetti+ MNPs and corresponding Monte Carlo–simulated data were determined and subsequently used for calculation of the division event (Supplementary Figure S5). Under these conditions, the number of Confetti+ MNP division events in the cortex of mice with NTN was about 3; and in the medulla of mice under an HAD, it was about 2.5 (Figure 2g–i). However, some Confetti+ clones were also seen in healthy controls, presumably due to homeostatic proliferation of tissue-resident MNPs, as described.9Hashimoto D. Chow A. Noizat C. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar Nevertheless, this verified that many Confetti+ clones in both disease models were derived from tissue-resident MNPs. To determine whether the Confetti+ clones were of the M1 or M2 differentiation type, we stained tissue sections for CD86 and CD206 (Supplementary Figure S6), respectively. We found that ≈25% of the Confetti+ clones in NTN contained most cells expressing one of these markers (Figure 2j). In HAD mice, there were more CD86+ M1 compared with CD206+ M2 Confetti clones (Figure 2j). For further phenotypic characterization, we also ascertained that Confetti+ clones in both, NTN and HAD, were F4/80+ (Figure 1), CD11b+, major histocompatibility complex II+, and CD11c+ (Supplementary Figure S6), consistent with tissue-resident MNPs. Finally, we wanted to validate the local proliferation of MNP clones. To this end, we labeled proliferating cells by injecting BrdU 5 and 6 days after NTN or 8 and 9 days after HAD induction and analyzed their kidneys for BrdU+ cells. Flow cytometric analysis revealed that ≈35% of the Confetti+ MNPs from NTN mice and 20% from HAD mice were BrdU+ (Figure 2k; Supplementary Figure S7). To detect proliferation in clones expressing the Confetti labels, we performed immunofluorescence microscopy using an anti-GFP antibody that detects GFP and yellow fluorescent protein, because the BrdU staining protocol for tissue sections destroys the Confetti label. Indeed, we detected Confetti+ clones containing BrdU+ cells (Figure 2l and m). Moreover, we confirmed the presence of BrdU+ cells also among clones of F4/80+ MNPs in kidney sections (Supplementary Figure S8), further supporting local MNP proliferation. Finally, we also observed a higher proportion of CD86+ M1 compared with CD206+ M2 cells in BrdU+ Confetti+ cells (Supplementary Figure S9), consistent with the histologic result. We here demonstrate that MNPs clonally expand in inflamed renal compartments during experimental glomerulonephritis and tubulointerstitial nephritis. Previous immunologic studies reported that tissue-resident MNPs can maintain their abundance by slow clonal proliferation in several uninflamed tissues of adult mice, including spleen, lung, and kidney, and in some studies, with a lower turnover rate, also microglia, Kupffer cells, and Langerhans cells.2Liu F. Dai S. Feng D. et al.Distinct fate, dynamics and niches of renal macrophages of bone marrow or embryonic origins.Nat Commun. 2020; 11: 2280Crossref PubMed Scopus (37) Google Scholar,5Masuda T. Amann L. Monaco G. et al.Specification of CNS macrophage subsets occurs postnatally in defined niches.Nature. 2022; 604: 740-748Crossref PubMed Scopus (37) Google Scholar,6Tay T.L. Mai D. Dautzenberg J. et al.A new fate mapping system reveals context-dependent random or clonal expansion of microglia.Nat Neurosci. 2017; 20: 793-803Crossref PubMed Scopus (345) Google Scholar,9Hashimoto D. Chow A. Noizat C. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar The speed of this steady-state turnover was in the order of 8, 12, or 16 weeks or longer, depending on the labeling system used. This may explain why we failed to detect clonal expansion of renal MNPs in healthy mice after only 14 to 17 days. A previous study had used Microfetti mice to show that microglia proliferated within 1 week after brain injury after unilateral facial nerve axotomy, compared with only once within 8 weeks under homeostatic conditions.6Tay T.L. Mai D. Dautzenberg J. et al.A new fate mapping system reveals context-dependent random or clonal expansion of microglia.Nat Neurosci. 2017; 20: 793-803Crossref PubMed Scopus (345) Google Scholar This suggested that tissue MNPs might clonally proliferate under inflammatory conditions also in other organs. Indeed, we here found that also kidney MNPs clonally expanded in situ after 1 week. These renal MNPs were mostly F4/80+ CD11b+ CD11c+ major histocompatibility complex II+, consistent with tissue-resident MNPs, previously classified as both dendritic cells and macrophages.1Kurts C. Panzer U. Anders H.J. et al.The immune system and kidney disease: basic concepts and clinical implications.Nat Rev Immunol. 2013; 13: 738-753Crossref PubMed Scopus (460) Google Scholar More important, 8 weeks after labeling, when hardly any Confetti+ monocytes remained, almost as many intrarenal Confetti+ clones were detected in both disease models. Some of these clones presumably had resulted from homeostatic proliferation,9Hashimoto D. Chow A. Noizat C. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar implying that some Confetti+ clones 1 week after labeling had originated from recruited monocytes. Nevertheless, these findings verified that many of the locally proliferating MNP clones had originated from tissue-resident cells. Our findings show that the accumulation of disease-driving MNPs during nephritis partially results from clonal in situ proliferation at sites of inflammation. Although the NTN and HAD models are imperfect mimics of human crescentic glomerulonephritis or acute interstitial nephritis, these findings suggest that therapeutic strategies to treat nephritis solely by preventing the recruitment of circulating monocytes may be of limited effectiveness, because MNPs may still accumulate through local proliferation. All the authors declared no competing interests. We acknowledge support by the Microscopy and the Flow Cytometry Core Facility and the central animal facilities of the Medical Faculty of Bonn University. We thank our technician Melanie Eichler for the excellent technical support. This work was supported by the German Research Foundation (DFG) through SFB1192 number 264599542, IRTG2168 number 272482170, TRR237 number 369799452, KFO329 number 386793560, SFB1454 number 432325352, and EXC2151 number 390873048 and by a Gottfried Wilhelm Leibniz Award to CK. JY and CK conceptualized the study and wrote the article. JY performed the experiments and the bioinformatic analysis. JL helped with the bioinformatic analysis. MP, UP, ZA, and MQ provided essential tools and know-how. All authors discussed and interpreted the data. Download .pdf (9.0 MB) Help with pdf files Supplementary File (PDF) Supplementary Methods. Supplementary Figure S1. Multichannel micrograph of Confetti+ mononuclear phagocytes (MNPs) in the kidney of Microfetti mice. Supplementary Figure S2. Location of cellular compartments of Confetti-labeled mononuclear phagocytes (MNPs). Supplementary Figure S3. Rendering model of confocal stack image of kidney from Microfetti mice. Supplementary Figure S4. Flow cytometry analysis of Confetti labeling in Ly6c+ blood monocytes and F4/80+ renal mononuclear phagocytes (MNPs). Supplementary Figure S5. Rendering model (top row) of confocal stack image of kidney from healthy and nephrotoxic nephritis (NTN) mice. Supplementary Figure S6. Confocal images showing the expression of surface markers on Confetti-labeled mononuclear phagocytes (MNPs). Supplementary Figure S7. Gating strategy of BrdU+ cells (Figure 2k) among Confetti+ cells in kidneys of Microfetti mice. Supplementary Figure S8. Confocal images of BrdU+ F4/80+ mononuclear phagocytes (MNPs) in kidneys from nephrotoxic nephritis (NTN)– and high adenine diet (HAD)–treated Microfetti mice. Supplementary Figure S9. Cell proportion of BrdU+ CD86+ M1 and CD206+ M2 cells within Confetti+ BrdU+ cells from nephrotoxic nephritis (NTN)– and high adenine diet (HAD)–treated Microfetti mice. Supplementary References.